Wearing contact lenses has been identified as a risk factor for the development of eye conditions such as giant papillary conjunctivitis and keratitis. We hypothesized that wearing contact lenses is associated with changes in the ocular microbiota. We compared the bacterial communities of the conjunctiva and skin under the eye from 58 subjects and analyzed samples from 20 subjects (9 lens wearers and 11 non-lens wearers) taken at 3 time points using a 16S rRNA gene-based sequencing technique (V4 region; Illumina MiSeq). We found that using anesthetic eye drops before sampling decreases the detected ocular microbiota diversity. Compared to those from non-lens wearers, dry conjunctival swabs from lens wearers had more variable and skin-like bacterial community structures (UniFrac; P value = 3.0). The results indicate that wearing contact lenses alters the microbial structure of the ocular conjunctiva, making it more similar to that of the skin microbiota. Further research is needed to determine whether the microbiome structure provides less protection from ocular infections. IMPORTANCE As in other body sites (i.e., the gut, skin, and mouth), the eye has a normal community of bacteria which are expected to confer resistance that provides protection from invaders. However, the eye microbiome has been largely neglected and is relevant to eye health and understanding eye diseases and to discovery of its functions. This report of a baseline study shows differences in the eye microbiome of contact lens wearers in relation to those of non-lens wearers and has the potential to help future studies explore novel insights into a possible role of the microbiome in the increased risk for eye infections in contact lens wearers.

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae . In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10 ⁻⁸ ml/min and 1.91 × 10 ⁻⁸ ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10 ⁻² CFU/ml for S. Typhimurium and 4.58 × 10 ⁻⁵ CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.

Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B₁₂ uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.

Newborns delivered by C-section acquire human skin microbes just after birth, but the sources remain unknown. We hypothesized that the operating room (OR) environment contains human skin bacteria that could be seeding C-section born infants. To test this hypothesis, we sampled 11 sites in four operating rooms from three hospitals in two cities. Following a C-section procedure, we swabbed OR floors, walls, ventilation grids, armrests, and lamps. We sequenced the V4 region of the 16S rRNA gene of 44 samples using Illumina MiSeq platform. Sequences were analyzed using the QIIME pipeline. Only 68 % of the samples (30/44, >1000 sequences per site) yielded sufficient DNA reads to be analyzed. The bacterial content of OR dust corresponded to human skin bacteria, with dominance of Staphylococcus and Corynebacterium. Diversity of bacteria was the highest in the ventilation grids and walls but was also present on top of the surgery lamps. Beta diversity analyses showed OR dust bacterial content clustering first by city and then by hospital (t test using unweighted UniFrac distances, p < 0.05). We conclude that the dust from ORs, collected right after a C-section procedure, contains deposits of human skin bacteria. The OR microbiota is the first environment for C-section newborns, and OR microbes might be seeding the microbiome in these babies. Further studies are required to identify how this OR microbiome exposure contributes to the seeding of the neonatal microbiome. The results might be relevant to infant health, if the current increase in risk of immune and metabolic diseases in industrialized societies is related to lack of natural exposure to the vaginal microbiome during labor and birth.

Background: Bacillus cereus is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. The incidence of B. cereus food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against B. cereus need to be developed. Endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents. Results: The endolysin from B. cereus phage B4, designated LysB4, was identified and characterized. In silico analysis revealed that this endolysin had the VanY domain at the N terminus as the catalytic domain, and the SH3_5 domain at the C terminus that appears to be the cell wall binding domain. Biochemical characterization of LysB4 enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at pH 8.0-10.0 and 50°C. The lytic activity was dependent on divalent metal ions, especially Zn²⁺. The antimicrobial spectrum was relatively broad because LysB4 lysed Gram-positive bacteria such as B. cereus, Bacillus subtilis and Listeria monocytogenes and some Gram-negative bacteria when treated with EDTA. LC-MS analysis of the cell wall cleavage products showed that LysB4 was an L-alanoyl-D-glutamate endopeptidase, making LysB4 the first characterized endopeptidase of this type to target B. cereus. Conclusions: LysB4 is believed to be the first reported L-alanoyl-D-glutamate endopeptidase from B. cereus-infecting bacteriophages. The properties of LysB4 showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes LysB4 a good candidate as a biocontrol agent against B. cereus and other pathogenic bacteria.

Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from 24°C to 65°C). It showed maximal activity at 50°C, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.